Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells

ABSTRACT

A chimeric LL2 monoclonal antibody is described in which the complementarity determining regions (CDRs) of the light and heavy chains of the murine LL2 anti-B-lymphoma, anti-leukemia cell monoclonal antibody has been recombinantly joined to the human kappa and IgG 1  constant region domains, respectively, which retains the immunospecificity and B-cell lymphoma and leukemia cell internalization capacity of the parental murine LL2 monoclonal antibody, and which has the potential of exhibiting reduced human anti-mouse antibody production activity. A humanized LL2 monoclonal antibody is described in which the CDRs of the light and heavy chains have been recombinantly joined to a framework sequence of human light and heavy chains variable regions, respectively, and subsequently linked to human kappa and IgG 1  constant region domains, respectively, which retains the immunospecificity and B-lymphoma and leukemia cell internalization capacities of the parental murine and chimeric LL2 monoclonal antibodies, and which has the potential for exhibiting reduced human anti-mouse antibody production activity. Vectors for producing recombinant chimeric and humanized chimeric monoclonal antibodies are provided. Isolated DNAs encoding the amino acid sequences of the LL2 variable light and heavy chain and CDR framework regions are described. Conjugates of chimeric and humanized chimeric LL2 antibodies with cytotoxic agents or labels find use in therapy and diagnosis of B-cell lymphomas and leukemias.

This is a continuation of U.S. Ser. No. 08/289,576, filed Aug. 12, 1994,now abandoned

BACKGROUND OF THE INVENTION

The invention relates generally to immunoconjugates for diagnostic andtherapeutic uses in cancer. In particular, the invention relates torecombinantly produced chimeric and humanized monoclonal antibodiesdirected against B-cell lymphoma and leukemia cells, which antibodiescan be covalently conjugated to a diagnostic or therapeutic reagentwithout loss of antibody binding and internalization function and withreduced production of human anti-mouse antibodies.

Non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia are B-cellmalignancies that remain important contributors to cancer mortality. Theresponse of these malignancies to various forms of treatment is mixed.They respond reasonably well to chemotherapy, and, in cases whereadequate clinical staging of NHL is possible, as for patients withlocalized disease, satisfactory treatment may be provided using fieldradiation therapy (Hall et al., Radiology for the Radiologist,Lippincott, Pa., 1989, pp 365-376). However, the toxic side effectsassociated with chemotherapy and the toxicity to the hematopoieticsystem from local, as well as whole body, radiotherapy, limits the useof these therapeutic methods. About one-half of the patients die fromthe disease (Posner et al., Blood, 61:705 (1983)).

The use of targeting monoclonal antibodies conjugated to radionuclidesor other cytotoxic agents offers the possibility of delivering suchagents directly to the tumor site, thereby limiting the exposure ofnormal tissues to toxic agents (Goldenberg, Semin. Nucl. Med., 19:332(1989)). In recent years, the potential of antibody-based therapy andits accuracy in the localization of tumor-associated antigens have beendemonstrated both in the laboratory and clinical studies (see., e.g.,Thorpe, TIBTECH, 11:42 (1993); Goldenberg, Scientific American, Science& Medicine, 1:64 (1994); Baldwin et al., U.S. Pat. No. 4,925,922 and4,916,213; Young, U.S. Pat. No. 4,918,163; U.S. Pat. No. 5,204,095; Irieet al., U.S. Pat. No. 5,196,337; Hellstrom et al., U.S. Pat. No.5,134,075 and 5,171,665). In general, the use of radio-labeledantibodies or antibody fragments against tumor-associated markers forlocalization of tumors has been more successful than for therapy, inpart because antibody uptake by the tumor is generally low, ranging fromonly 0.01% to 0.001% of the total dose injected (Vaughan et al., Brit.J. Radiol., 60:567 (1987)). Increasing the concentration of theradiolabel to increase the dosage to the tumor is counterproductivegenerally as this also increases exposure of healthy tissue toradioactivity.

LL-2 (EPB2) is a highly specific anti-B-cell lymphoma andanti-lymphocytic leukemia cell murine monoclonal antibody (mAb) that israpidly internalized by such cells and that can overcome some of theaforementioned difficulties (Shih et al., Int. J. Cancer, 56:538(1994)). LL2, which is of the IgG2a antibody type, was developed usingthe Raji B-lymphoma cell line as the source of antigen(Pawlak-Byczkowska et al., Cancer Res., 49:4568 (1989)). Murine LL2(mLL2) is known to react with an epitope of CD22 (Belisle et al., ProcAmer. Assn. Clin. Res., 34:A2873 (1993)). CD22 molecules are expressedin the cytoplasm of progenitor and early pre-B cells, and appear in thecell surface of mature B-cells.

By immunostaining of tissue sections, mLL2 was shown to react with 50 of51 B-cell lymphomas tested. mLL2 is a highly sensitive means ofdetecting B-cell lymphoma cell in vivo, as determined by aradioimmunodetection method (Murthy et al., Eur. J. Nucl. Med., 19:394(1992)). The Fab' fragment of mLL2 labeled with ^(99m) Tc localized to63 of 65 known lesions in Phase II trial patients with B-cell lymphoma(Mills et al., Proc. Amer. Assn. Cancer Res., 14:A2857 (1993)). Inaddition, ¹³¹ I-labeled mLL2 was therapeutically effective in B-celllymphoma patients (Goldenberg et al., J. Clin. Oncol., 9:548 (1991)).mLL2 Fab' conjugated to the exotoxin PE38KDEL induced completeremissions of measurable human lymphoma xenografts (CA-46) growing innude mice (Kreitman et al., Cancer Res., 53:819 (1993)).

The clinical use of mLL2, just as with most other promising murineantibodies, has been limited by the development in humans of a HAMAresponse. While a HAMA response is not invariably observed followinginjection of mLL2, in a significant number of cases patients developedHAMA following a single treatment with mLL2. This can limit thediagnostic and therapeutic usefulness of such antibody conjugates, notonly because of the potential anaphylactic problem, but also as a majorportion of the circulating conjugate may be complexed to and sequesteredby the circulating anti-mouse antibodies. This is exemplified by onestudy in which about 30% of the patients developed low level HAMAresponse following a single injection of about 6 mg of mLL2 ¹³¹ I-IgGand nearly all developed a strong HAMA response with additionalinjections. On the other hand, with mLL2 Fab' labeled with ^(99m) Tc, noHAMA response was observed. Such HAMA responses in general pose apotential obstacle to realizing the full diagnostic and therapeuticpotential of the mLL2 antibody.

Although, as noted above, the use of fragments of mLL2, such as F(ab')₂and Fab', partially alleviate/circumvent these problems ofimmunogenicity, there are circumstances in which whole IgG is moredesirable, such as when induction of cellular immunity is intended fortherapy, or where an antibody with enhanced survival time is required.

In order to maximize the value of the mLL2 IgG antibody as a therapeuticor diagnostic modality and increase its utility in multiple andcontinuous administration modalities, it is an object of this inventionto produce a mouse/human chimeric mAb (cLL2) and a humanized mAb (hLL2)related to mLL2 that retain the antigen-binding specificity of mLL2, butthat elicit reduced HAMA in a subject receiving same.

It is another object of this invention to provide DNA sequences encodingthe amino acid sequences of the variable regions of the light and heavychains of the cLL2 and hLL2 mobs, including the complementaritydetermining regions (CDR).

It is also an object of this invention provide conjugates of the hLL2and cLL2 mAbs containing therapeutic or diagnostic modalities.

It is a further object of this invention to provide methods of therapyand diagnosis that utilize the humanized and chimeric mAbs of theinvention.

These objects have been achieved by the invention described below in thespecification and appended claims.

SUMMARY OF THE INVENTION

In one aspect of the invention, there is provided a cLL2 mAb related tomLL2 mAb, in which the murine light (VK) and heavy (VH) chain variableregions are joined to the human constant light (kappa) and heavy (IgG₁)chains. This chimeric mAb retains the B-lymphoma and leukemia celltargeting and internalization properties of the parental mLL2.

In another aspect of the invention, there is provided a hLL2 mAb relatedto mLL2 mAb, in which the complementarity-determining regions (CDRs) ofthe light and heavy chains of the mLL2 mAb are joined to the framework(FR) sequence of human VK and VH regions, respectively, and subsequentlyto the human kappa and IgG₁ constant region domains, respectively. Thishumanized antibody retains the B-lymphoma and leukemia cell targetingand internalization characteristics of the parental mLL2 mAb, and canexhibit a lowered HAMA reaction.

In still another aspect, there is provided isolated polynucleotidescomprising DNA sequences encoding the amino acid sequences of thevariable light and heavy chains, respectively, of the hLL2 and cLL2mAbs.

In an additional aspect, there is provided the amino acid sequences ofthe CDRs of the VK and VH chains.

In yet another aspect, there are provided conjugates in which the hLL2or cLL2 mAb is covalently bonded to a diagnostic or therapeutic reagent.

In still another aspect, there are provided methods whereby theaforementioned mAb conjugates can be used to diagnose or treat B-celllymphomas and lymphocytic leukemias.

These and other aspects and embodiments of the invention will becomeapparent by reference to the following specification and appendedclaims.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a comparison of the amino acid sequences between murine LL2and humanized LL2REIHuVK (SEQ ID Nos. 2 and 6) and between murine LL2and humanized EUHuVH1 and EUHuVH2, and NEWMHuVH1 and NEWMHuVH2 (SEQ IDNos. 4, 9 and 8). Asterisks indicate murine LL2 sequences that aredifferent from that of the human FR at corresponding positions. CDRs areboxed. FR resides showing CDR contacts by computer modeling areunderlined.

FIG. 2A-2B show vicinal relationships of the LL2 CDRs to their frameworkregions (FRs). Separate energy-minimized models for the VK and VHdomains of mLL2 were constructed, and all FR residues within a radius of4.5 Å or any CDR atom were identified as potential CDR-FR contacts. CDRsof the light (L1, L2, and L3, FIG. 2A) and heavy (H1, H2, and H3, FIG.2B)) chains are shown as "ball and stick" representations superimposedon their respective, space-filling FRs.

FIG. 3A shows the light chain staging (VKpBR) and mammalian expression(pKH) vectors, while FIG. 3B shows the staging (VHpBS) and mammalianexpression (pG1g) vectors.

FIGS. 4A-4B show the double-stranded DNA and amino acid sequences of theLL2 VK domain (FIG. 4A, SEQ ID NOS. 1 and 2) and the LL2 VH domain (FIG.4B, SEQ ID NOS. 3 and 4). Amino acid sequences encoded by thecorresponding DNA sequences are given as one letter codes. CDR aminoacid sequences are boxed. The Asn-glycosylation site located in FR1 ofLL2VK (FIG. 4A, SEQ ID NO. 2) is shown as the underlined NVT sequence.

FIG. 5A shows the double stranded DNA and corresponding amino acidresidues of the hLL2 VK domain (SEQ ID NOS. 5 and 6). CDR amino acidsequences are boxed. The corresponding data for the VH domain is shownin FIG. 5B (SEQ ID NOS. 7 and 8).

FIG. 6 is a schematic diagram representation of the PCR/gene synthesisof the humanized VH region and the subcloning into the staging vector,VHpBS.

FIG. 7 shows SDS-PAGE analysis of mLL2 and cLL2 antibodies undernon-reducing (lanes 6-8) and reducing (lanes 3-5, light and heavychains) conditions. Lanes 3 and 6 include a control antibody.

FIG. 8 shows SDS-PAGE analysis of different versions of cLL2 and hLL2antibodies under reducing (lanes 3-5) and non-reducing (lanes 6-8)conditions.

FIG. 9 shows SDS-PAGE analysis on mix-and-match cLL2 and hLL2 antibodiesunder reducing (lanes 3-6) and non-reducing (lanes 7-10) conditions.cLL2 serves as the control.

FIG. 10 shows the results of a comparative Raji cell competitiveantibody binding assay involving mLL2 and cLL2 antibodies competing forbinding to cells against tracer radiolabeled mLL2.

FIG. 11A shows the results of a comparative Raji cell competitiveantibody binding assay in which mixed humanized/chimeric LL2s werecompared to cLL2, while FIG. 11B shows a similar comparison with twoversions of hLL2 compared to cLL2.

FIG. 12 shows a comparison of antibody internalization:surface bindingratios as a function of time for cLL2, cLL2 (Q to V mutagenesis), hLL2and mLL2 antibodies.

FIG. 13 shows an SDS-PAGE analysis of mLL2 and cLL2 afterdeglycosylation by endoglycosidase F.

FIG. 14 shows the effect of deglycosylation of mLL2 on its bindingaffinity to Raji cells.

DETAILED DESCRIPTION OF THE INVENTION

cDNAs encoding the VL and VH regions of the mLL2 mAb have been isolatedand separately recombinantly subcloned into mammalian expression vectorscontaining the genes encoding kappa and IgG₁ constant regions,respectively, of human antibodies. Cotransfection of mammalian cellswith these two recombinant DNAs expressed a cLL2 mAb that, like theparent mLL2 mAb, bound avidly to, and was rapidly internalized by,B-lymphoma cells.

The CDRs of the VK and VH DNAs have been similarly recombinantly linkedto the framework (FR) sequences of the human VK and VH regions,respectively, which are subsequently linked, respectively, to the humankappa and IgG₁ constant regions, so as to express in mammalian cells asdescribed above hLL2.

In this specification, the expressions "cLL2" or "cLL2 mAb" are intendedto refer to the chimeric monoclonal antibody constructed by joining orsubcloning the murine VK and VH regions to the human constant light andheavy chains, respectively. The expressions "hLL2" or "hLL2 mAb" areintended to refer to the humanization of the chimeric monoclonalantibody by replacing the murine FR sequences in cLL2 with that of humanframework regions.

Covalent conjugates between cLL2 and hLL2 mAbs and a diagnostic orchemotherapeutic reagent, formulated in pharmaceutically acceptablevehicles (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., MackPublishing Co., Easton, Pa., 1990) can be prepared that have theadvantages, compared to prior art antibody conjugates, of B-celllymphoma-specific and leukemia cell-specific targeting, rapidinternalization into target cells, rapid liberation of the diagnostic orchemotherapeutic reagent intracellularly (thereby increasingeffectiveness of the reagent), and a potential reduction of the HAMAresponse in the human patient.

As the VK-appended carbohydrate moiety of the cLL2 mAb is shown hereinnot to be involved in binding to B-lymphoma cells, it is preferred touse conjugates in which the reagent is bound to the antibody throughsuch carbohydrate moieties, such as through oxidized carbohydratederivatives. Methods for the production of such conjugates and their usein diagnostics and therapeutics are provided, for example, in Shih etal., U.S. Pat. No. 5,057,313, Shih et al., Int. J. Cancer 41:832 (1988),and copending, commonly owned Hansen et al., U.S. Ser. No. 08/162,912,the contents of which are incorporated herein by reference. Directlinkage of the reagent to oxidized carbohydrate without the use of apolymeric carrier is described in McKearn et al., U.S. Pat. No.5,156,840, which is also incorporated by reference.

A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the antibodies of the invention. Theseinclude: chemotherapeutic drugs such as doxorubicin, methotrexate,taxol, and the like; chelators, such as DTPA, to which detectable labelssuch as fluorescent molecules or cytotoxic agents such as heavy metalsor radionuclides can be complexed; and toxins such as Pseudomonasexotoxin, and the like. Several embodiments of these conjugates aredescribed in the examples below.

Cell lines and culture media used in the present invention include ILL2(EPB-2) hybridoma cells (Pawlak-Byczkowska et al. 1989 above),Sp2/0-Ag14 myeloma cells (ATCC, Rockville, Md.) and Raji cells. Thesecells are preferably cultured in Dulbecco's modified Eagle's Medium(DMEM) supplemented with 10% FCS (Gibco/BRL, Gaithersburg, Mass.), 2 mML-glutamine and 75 μg/ml gentamicin, (complete DMEM). Transfectomas aregrown in Hybridoma Serum Free Medium, HSFM, (Gibco/BRL, Gaithersburg,Mass.) containing 10% of FCS and 75 μg/ml gentamicin (complete HSFM) or,where indicated, in HSFM containing only antibiotics. Selection of thetransfectomas may be carried out in complete HSFM containing 500 μg/mlof hygromycin (Calbiochem, San Diego, Calif.). All cell lines arepreferably maintained at 37° C. in 5% CO₂.

An important aspect of this invention is that antibody variable domainscan be modeled by computer modeling (see, for example, Dion, inGoldenberg et al. eds., Cancer Therapy With Radiolabeled Antibodies, CRCPress, Boca Raton, Fla., 1994), which is incorporated by reference. Ingeneral, the 3-D structure for both the mLL22 and hLL2 mAbs are bestmodeled by homology. The high frequency of residue identities (75.0 to92.3%) between the deduced primary sequences of mLL2 light chain FRregions and human REI (VK) facilitates this approach because of theavailability of crystallographic data from the Protein Data Bank (PDRCode 1REI, Bernstein et al., J. Mol. Biol. 112:535 (1977)), which isincorporated by reference. Similarly, antibody EU (VH) sequences can beselected as the computer counterparts for FR1 to FR3 of the mLL2 heavychain; FR4 was based on NEWM. As X-ray coordinate data is currentlylacking for the EU sequence, NEWM structural data (PDR Code 3FAB) forFRs 1 to 4 can be used, and amino acid side groups can be replaced tocorrespond to mLL2 or EU (hLL2) as needed. The CDR of the light chaincan be modeled from the corresponding sequence of 1MCP (L1 and L2) and1REI (L3). For heavy chain CDRs, H1 and H2 can be based on 2HFL and1MCP, respectively, while H3 can be modeled de novo. Wherever possible,side group replacements should be performed so as to maintain thetorsion angle between Cα and Cβ. Energy minimization may be accomplishedby the AMBER forcefield (Weiner et al, J. Amer. Chem. Soc. 106:765(1984) using the convergent method. Potentially critical FR-CDRinteractions can be determined by initially modeling the light and heavyvariable chains of mLL2. All FR residues within a 4.5 Å radius of allatoms within each CDR can thereby be identified and retained in thefinal design model of hLL2.

Once the sequences for the hLL2 VK and VH domains are designed, CDRengrafting can be accomplished by gene synthesis using long syntheticDNA oligonucleotides as templates and short oligonucleotides as primersin a PCR reaction. In most cases, the DNA encoding the VK or VH domainwill be approximately 350 bp long. By taking advantage of codondegeneracy, a unique restriction site may easily be introduced, withoutchanging the encoded amino acids, at regions close to the middle of theV gene DNA sequence. For example, at DNA nucleotide positions 157-162(amino acid positions 53 and 54) for the hLL2 VH domain, a unique AvrIIsite can be introduced while maintaining the originally designed aminoacid sequence (FIG. 4B). Two long non-overlapping single-stranded DNAoligonucleotides (˜150 bp) upstream and downstream of the AvrII site(see, for example, oligo A and oligo B, Example 3 below) can begenerated by automated DNA oligonucleotide synthesizer (Cyclone Plus DNASynthesizer, Milligen-Biosearch). As the yields of full length DNAoligonucleotides such as oligos A and B may be expected to be low, theycan be amplified by two pairs of flanking oligonucleotides (oligo Seq.ID Nos. 10 and 11 for oligo A; oligo Seq. ID Nos. 12 and 13 for oligo B,Example 3) in a PCR reaction. The primers can be designed with thenecessary restriction sites to facilitate subsequent subcloning. Primersfor oligo A and for oligo B should contain overlapping sequence at theAvrII site so that the resultant PCR product for oligo A and B,respectively, can be joined in-frame at the AvrII site to form a fulllength DNA sequence (ca 350 bp) encoding the hLL2 VH domain. Theligation of the PCR products for oligo A (restriction-digested with PstIand AvrII) and B (restriction-digested with AvrII and BstEII) at theAvrII site and their subcloning into the PstII/BstEII sites of thestaging vector, VHpBS, can be completed in a singlethree-fragment-ligation step (See, for example, Example 3). Thesubcloning of the correct sequence into VHpBS can be first analyzed byrestriction digestion analysis and subsequently confirmed by sequencingreaction according to Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463(1977).

The HindIII/BamHI fragment containing the Ig promoter, leader sequenceand the hLL2 VH sequence can be excised from the staging vector andsubcloned to the corresponding sites in a pSVgpt-based vector, pG1g,which contains the genomic sequence of the human IgG constant region, anIg enhancer and a gpt selection marker, forming the final expressionvector, hLL2pG1g. Similar strategies can be employed for theconstruction of the hLL2 VK sequence. The restriction site chosen forthe ligation of the PCR products for the long oligonucloetides (oligos Cand D, see examples below) can be NruI in this case.

The DNA sequence containing the Ig promoter, leader sequence and thehLL2 VK sequence can be excised from the staging vector VKpBR bytreatment with BamH1/HindIII, and can be subcloned into thecorresponding sites of a pSVhyg-based vector, pKh, which contains thegenomic sequence of human kappa chain constant regions, a hygromycinselection marker, an Ig and a kappa enhancer, forming the finalexpression vector, hLL2pKh.

As humanization sometimes results in a reduction or even loss ofantibody affinity, additional modification might be required in order torestore the original affinity (See, for example, Tempest et al.,Bio/Technology 9:266 (1991); Verhoeyen et al., Science 239:1534 (1988)),which are incorporated by reference. Knowing that cLL2 exhibits abinding affinity comparable to that of its murine counterpart (seeExample 5 below), defective designs, if any, in the original version ofhLL2 can be identified by mixing and matching the light and heavy chainsof cLL2 to those of the humanized version. SDS-PAGE analysis of thedifferent mix-and-match humanized chimeric LL2 under non-reducing (thedisulfide L-H chain connections remain intact) and reducing conditions(the chains separate, permitting analyses of the relationships of thedifferent types of light and heavy chains on the properties of themolecule). For example, migration as multiple bands or as a higherapparent molecular size can be due to the presence of a glycan group atthe N-linked glycosylation site found at the FR1 region of the murine VKdomain of LL2. For another example, a discrete band migrating at about25 kDa is the expected molecular size for a non-glycosylated lightchain.

In general, to prepare cLL2 mAb, VH and VK chains of mLL2 can beobtained by PCR cloning using DNA products and primers. Orlandi et al.,infra, and Leung et al., infra. The VK PCR primers may be subcloned intoa pBR327 based staging vector (VKpBR) as described above. The VH PCRproducts may be subcloned into a similar pbluescript-based stagingvector (VHpBS) as described above. The fragments containing the VK andVH sequences, along with the promoter and signal peptide sequences, canbe excised from the staging vectors using HindIII and BamHI restrictionendonucleases. The VK fragments (about 600 bp) can be subcloned into amammalian expression vector (for example, pKh) conventionally. pKh is apSVhyg-based expression vector containing the genomic sequence of thehuman kappa constant region, an Ig enhancer, a kappa enhancer and thehygromucin-resistant gene. Similarly, the about 800 bp VH fragments canbe subcloned into pG1g, a pSVgpt-based expression vector carrying thegenomic sequence of the human IgG1 constant region, an Ig enhancer andthe xanthine-guanine phosphoribosyl transferase (gpt) gene. The twoplasmids may be transfected into mammalian expression cells, such asSp2/0-Ag14 cells, by electroporation and selected for hygromycinresistance. Colonies surviving selection are expanded, and supernatantfluids monitored for production of cLL2 mAb by an ELISA method. Atransfection efficiency of about 1-10×10⁶ cells is desirable. Anantibody expression level of between 0.10 and 2.5 μg/ml can be expectedwith this system.

RNA isolation, cDNA synthesis, and amplification can be carried out asfollows. Total cell RNA can be prepared from a LL2 hybridoma cell line,using a total of about 10⁷ cells, according to Sambrook et al.,(Molecular Cloning: A Laboratory Manual, Second ed., Cold Spring HarborPress, 1989), which is incorporated by reference. First strand cDNA canbe reverse transcribed from total RNA conventionally, such as by usingthe SuperScript preamplification system (Gibco/BRL., Gaithersburg, Md.).Briefly, in a reaction volume of 20 μl, 50 ng of random primers can beannealed to 5 μg of RNAs in the presence of 2 μl of 10× synthesis buffer200 mM Tris-HCl (pH 8.4), 500 mM KCl, 25 mM MgCl₂, 1 mg/ml BSA!, 1 μl of10 mM dNTP mix, 2 μl of 0.1M DTT, and 200 units of SuperScript reversetranscriptase. The elongation step is initially allowed to proceed atroom temperature for 10 min followed by incubation at 42° C. for 50 min.The reaction can be terminated by heating the reaction mixture at. 90°C. for 5 min.

The VK and VH sequences for cLL2 or hLL2 can amplified by PCR asdescribed by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86:3833(1989)) which is incorporated by reference. VK sequences may beamplified using the primers CK3BH and VK5-3 (Leung et al.,BioTechniques, 15:286 (1993), which is incorporated by reference), whileVH sequences can be amplified using the primer CH1B which anneals to theCH1 region of murine 1gG, and VHIBACK (Orlandi et al., 1989 above). ThePCR reaction mixtures containing 10 μl of the first strand cDNA product,9 μl of 10×PCR buffer 500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl2,and 0.01% (w/v) gelatin! (Perkin Elmer Cetus, Norwalk, Conn.), can besubjected to 30 cycles of PCR. Each PCR cycle preferably consists ofdenaturation at 94° C. for 1 min, annealing at 50° C. for 1.5 min, andpolymerization at 72° C. for 1.5 min. Amplified VK and VH fragments canbe purified on 2% agarose (BioRad, Richmond, Calif.). See Example 3 fora method for the synthesis of an oligo A (149-mer) and an oligo B(140-mer) on an automated Cyclone Plus DNA synthesizer(Milligan-Biosearch) for use in constructing humanized V genes.

PCR products for VK can be subcloned into a staging vector, such as apBR327-based staging vector VKpBR that contains an Ig promoter, a signalpeptide sequence and convenient restriction sites to facilitate in-frameligation of the VK PCR products. PCR products for VH can be subclonedinto a similar staging vector, such as the pBluescript-based VHpBS.Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al., Proc. Natl.Acad. Sci., USA, 74:5463 (1977) which is incorporated by reference.

The DNA sequences described herein are to be taken as including allalleles, mutants and variants thereof, whether occurring naturally orinduced.

The two plasmids can be co-transfected into an appropriate cell, e.g.,myeloma Sp2/0-Ag14, colonies selected for hygromycin resistance, andsupernatant fluids monitored for production of cLL2 or hLL2 antibodiesby, for example, an ELISA assay, as described below.

Transfection, and assay for antibody secreting clones by ELISA, can becarried out as follows. About 10 μg of hLL2pKh (light chain expressionvector) and 20 μg of hLL2pG1g (heavy chain expression vector) can beused for the transfection of 5×10⁶ SP2/0 myeloma cells byelectroporation (BioRad, Richmond, Calif.) according to Co et al., J.Immunol., 148:1149 (1992) which is incorporated by reference. Followingtransfection, cells may be grown in 96-well microtiter plates incomplete HSFM medium (GIBCO, Gaithersburg, Md.) at 37° C., 5% CO₂. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 μg/ml of hygromycin. Colonies typically emerge 2-3weeks post-electroporation. The cultures can then be expanded forfurther analysis.

Transfectoma clones that are positive for the secretion of chimeric orhumanized heavy chain can be identified by ELISA assay. Briefly,supernatant samples (100 μl) from transfectoma cultures are added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH)-IgG, F(ab')₂ fragment-specific antibody (Jackson ImmunoResearch,West Grove, Pa.). Plates are incubated for 1 h at room temperature.Unbound proteins are removed by washing three times with wash buffer(PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP)conjugated GAH-IgG, Fc fragment-specific antibodies (JacksonImmunoResearch, West Grove, Pa.) are added to the wells, (100 μl ofantibody stock diluted×10⁴, supplemented with the unconjugated antibodyto a final concentration of 1.0 μg/ml). Following an incubation of 1 h,the plates are washed, typically three times. A reaction solution, 100μl, containing 167 μg of orthophenylene-diamine (OPD) (Sigma, St. Louis,Mo.), 0.025% hydrogen peroxide in PBS!, is added to the wells. Color isallowed to develop in the dark for 30 minutes. The reaction is stoppedby the addition of 50 μl of 4N HCl solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader (Bio-Tekinstruments, Winooski, Vt.). Bound chimeric antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 micron membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3M Tris (pH 8.6), and protein concentrationsdetermined from the absorbancies at 280/260 nm. Peak fractions arepooled, dialyzed against PBS, and the antibody concentrated, forexample, with the Centricon 30 (Amicon, Beverly, Mass.). The antibodyconcentration is determined by ELISA, as before, and its concentrationadjusted to about 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), isconveniently added to the sample as preservative.

Comparative binding affinities of the mLL2, cLL2 and hcLL2 antibodiesthus isolated may be determined by direct radioimmunoassay. mLL2 can belabeled with ¹³¹ I or ¹²⁵ I using the chloramine T method (see, forexample, Greenwood et al., Biochem. J., 89:123 (1963) which isincorporated by reference). The specific activity of the iodinatedantibody is typically adjusted to about 10 μCi/μg. Unlabeled and labeledantibodies are diluted to the appropriate concentrations using reactionmedium (HSFM supplemented with 1% horse serum and 100 μg/ml gentamicin).The appropriate concentrations of both labeled and unlabeled antibodiesare added together to the reaction tubes in a total volume of 100 μl. Aculture of Raji cells is sampled and the cell concentration determined.The culture is centrifuged and the collected cells washed once inreaction medium followed by resuspension in reaction medium to a finalconcentration of about 10⁷ cells/ml. All procedures are carried out inthe cold at 4° C. The cell suspension, 100 μl, is added to the reactiontubes. The reaction is carried out at 4° C. for 2 h with periodic gentleshaking of the reaction tubes to resuspend the cells. Following thereaction period, 5 ml of wash buffer (PBS containing 1% BSA) is added toeach tube. The suspension is centrifuged and the cell pellet washed asecond time with another 5 ml of wash buffer. Following centrifugation,the amount of remaining radioactivity remaining in the cell pellet isdetermined in a gamma counter (Minaxi, Packard Instruments, Sterling,Va).

The Raji cell surface antigen binding affinities of mix-and-match andfully humanized antibodies can be compared to that of cLL2 using variousconcentrations of mLL2 F(ab')₂ fragments devoid of the Fc portion ascompetitors, as evaluated by flow cytometry assay. Residualsurface-bound LL2 antibodies carrying the human Fc portions (cLL2 andmix-and-match LL2) can be detected by a FITC-labeled anti-human Fcspecific antibody in a flow cytometry assay. Where mix-and-match LL2antibodies exhibit antigen-binding affinities similar to that of cLL2,it can be concluded that the original designs for the humanization ofboth the light and heavy chains retain the mLL2 immunoreactivity.

The internalization of mLL2, cLL2 and hLL2 antibodies into target cellscan be followed by fluorescence labeling, essentially according to theprocedure of Pirker et al., J. Clin. Invest., 76:1261 (1985), which isincorporated by reference. Cultured Raji cells are centrifuged and thecells resuspended in fresh medium to a concentration of about 5×10⁶cells/ml. To each well of a 96-well microtiter plate, 100 μi of the cellsuspension is added. The antibodies, 40 μg/ml, in a volume of 100 μl areadded to the reaction wells at timed intervals so as to terminate allreactions simultaneously. The plate is incubated at 37° C. in a CO₂ cellculture incubator. Unbound antibodies are removed by washing the cellsthree times with cold 1% FCS/PBS at the end of the incubation. The cellsare then treated with 1 ml of Formaid-Fresh 10% formalin solution(Fisher, Fair Lawn, N.J.)! for 15 min at 4° C. After washing, antibodiespresent either on the cell surface or inside the cells are detected bytreatment with FITC-labeled goat anti-mouse antibody (Tago, Burlingame,Calif.), or FITC-labeled goat anti-human antibody (JacksonImmunoResearch, West Grove, Pa.), depending on whether the antibodybeing assayed for is murine, chimeric, or humanized, respectively.Fluorescence distributions are evaluated using a BH-2 fluorescencemicroscope (Olympus, Lake Success, N.Y.).

The rate of antibody internalization can be determined according toOpresko et al., (J. Biol. Chem., 262:4116 (1987)), using radioiodinatedantibody as tracer. Briefly, radiolabeled antibodies (1×10⁴ cpm) areincubated with the Raji cells (1×10⁶ cells/ml) at 4° C. for 2 h in 0.5ml of DMEM medium containing 1% human serum. Following the reactioninterval, non-specifically bound antibodies are removed by washing threetimes with 0.5 ml of DMEM medium. To each of the reaction tubes 0.5 mlof DMEM medium is added and the suspension incubated at 37° C. for thedetermination of internalization. At timed intervals, triplicates ofcells are removed and chilled immediately in an ice bath to stop furtherinternalization. Cells are centrifuged at 1000×g for 5 min at 4° C. Thesupernatant is removed and counted for radioactivity. The surface-boundradioactivity is removed by treatment with 1 ml 0.1M acetate/0.1Mglycine buffer at pH 3.0 for 8 min. in the cold. Radioactivity removedby the acid treatment, and that remaining associated with the cells, aredetermined. The ratio of the CPM_(internalization) /CPM_(surface) isplotted versus time to determine the rate of internalization from theslope.

Detailed protocols for oligonucleotide-directed mutagenesis and relatedtechniques for mutagenesis of cloned DNA are well-known. For example,see Sambrook et al. and Ausubel et al. above.

Asn-linked glycosylation sites may be introduced into antibodies usingconventional site-directed oligonucleotide mutagenesis reactions. Forexample, to introduce an Asn in position 18 of a kappa protein, one mayalter codon 18 from AGG to AAC. To accomplish this, a single strandedDNA template containing the antibody light chain sequence is preparedfrom a suitable strain of E. coli (e.g., dut⁻ ung-) in order to obtain aDNA molecule containing a small number of uracils in place of thymidine.Such a DNA template can be obtained by M13 cloning or by in vitrotranscription using a SP6 promoter. See, for example, Ausubel et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY,1987. An oligonucleotide containing the mutated sequence is synthesizedconventionally, annealed to the single-stranded template and the producttreated with T4 DNA polymerase and T4 DNA ligase to produce adouble-stranded DNA molecule. Transformation of wild type E. coli (dut⁺ung⁺) cells with the double-stranded DNA provides an efficient recoveryof mutated DNA.

Alternatively, an Asn-linked glycosylation site can be introduced intoan antibody light chain using an oligonucleotide containing the desiredmutation as the primer and DNA clones of the variable regions for the VLchain, or by using RNA from cells that produce the antibody of interestas a template. Also see, Huse, in ANTIBODY ENGINEERING: A PRACTICALGUIDE, Boerrebaeck, ed., W. H. Freeman & Co., pp 103-120, 1992.Site-directed mutagenesis can !be performed, for example, using theTRANSFORMER™ kit (Clontech, Palo Alto, Calif.) according to themanufacturer's instructions.

Alternatively, a glycosylation site can be introduced by synthesizing anantibody chain with mutually priming oligonucleotides, one suchcontaining the desired mutation. See, for example, Uhlmann, Gene 71:29(1988); Wosnick et al., Gene 60:115 (1988); Ausubel et al., above, whichare incorporated by reference.

Although the general description above referred to the introduction ofan Asn glycosylation site in position 18 of the light chain of anantibody, it will occur to the skilled artisan that it is possible tointroduce Asn-linked glycosylation sites elsewhere in the light chain,or even in the heavy chain variable region.

The representative embodiments described below are simply used toillustrate the invention. Those skilled in these arts will recognizethat variations of the present materials fall within the broad genericscope of the claimed invention. The contents of all references mentionedherein are incorporated by reference.

EXAMPLE 1 Choice of Human Frameworks and Sequence Design for theHumanization of LL2 Monoclonal Antibody

By comparing the murine variable (V) region framework (FR) sequences ofLL2 to that of human antibodies in the Kabat data base (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th ed., U.S.Department of Health and Human Services, U.S. Government PrintingOffice, Washington, D.C.), which is incorporated by reference, the humanREI (FIG. 1A, Sequence ID No. 6) and EU (FIG. 1B, Sequence ID NOS. 9 and8) sequences were found to exhibit the highest degree of sequencehomology to the FRs of VK and VH domains of LL2, respectively.Therefore, the REI and EU FRs were selected as the human frameworks ontowhich the CDRs for LL2 VK and VH were grafted, respectively. The FR4sequence of NEWM, however, rather than that of EU, was used to replacethe EU FR4 sequence for the humanization of LL2 heavy chain. Based onthe results of computer modeling studies (FIGS. 2A and 2B), murine FRresidues having potential CDR contacts, which might affect the affinityand specificity of the resultant antibody, were retained in the designof the humanized FR sequences (FIG. 1).

Two versions of humanized heavy chain were constructed. In the firstversion (hLL2-1, SEQ ID NO. 9), the glutamine (Q) at amino acid position5 (Kabat numbering) was introduced to include a PstI restriction site tofacilitate its subcloning into the staging vector (FIG. 3). This murineresidue was converted, by oligo-directed mutagenesis, to the human EUresidue valine (V) in hLL2-2SEQ ID NO. 8. It should be noted that in theoriginal murine kappa chain variable sequence, a potential N-linkedglycosylation site was identified at positions 18-20 (FIG. 1 SEQ ID NO:2) and was used for carbohydrate addition. This glycosylation site wasnot included in the REI FR sequence used for LL2 light chainhumanization.

See Example 3 for more oligonucleotide detail.

EXAMPLE 2 PCR Cloning and Sequence Elucidation for LL2 Heavy and LightChain Variable Regions

The variable regions for both heavy (VH) and light (VK) chains of mLL2(lgG2a) were obtained by PCR cloning using DNA primers as described ingeneral above and in greater detail in Example 3, below. As PCR is proneto mutations, the variable region sequence of multiple individual clonesfor either the heavy or light chains was determined for six clones andconfirmed to be identical prior to use for the construction of thechimeric antibody.

The PCR products for VK were subcloned into a pBR327-based stagingvector, VKpBR, which contained an Ig promoter, a signal peptide sequenceand convenient restriction sites to facilitate in-frame ligation of theVK PCR products (FIG. 3A). The PCR products for VH were subcloned into asimilar pBluescript-based staging vector, VHpBS (FIG. 3B).

As noted above, at least six individual clones containing the respectivePCR products were sequenced according to the method of Sanger et al.,1977, above. All were shown to bear identical sequences and theirrespective sequences were elucidated, as shown in FIG. 4A for LL2 VK(Sequence ID NO. 1) and in FIG. 4B for LL2 VH (Sequence ID NO. 3). Nodefective mutations were identified within the sequences encoding the VKand VH regions. Comparison of the PCR-amplified variable regionsequences of LL2 with the Kabat database (Kabat et al., above) suggestedthat the VK and VH sequences of LL2 belong to subgroup 5 and 2B,respectively. Important residues such as Cys for intra-domain disulfidelinkage were retained at appropriate positions.

In the FR1 framework region of VK, an N-linked carbohydrate attachmentsite, Asn-Val-Thr, was identified at position 18-20 (FIG. 4A, SEQ ID NO.2), suggesting that the VK of LL2 might be glycosylated. As will bedetailed below, SDS-PAGE analysis under reducing conditions demonstratedthat this Asn glycosylation site is indeed utilized for carbohydrateaddition. The presence of the glycosylation site in the variable regiondoes not, however, appear to affect the immunoreactivity of theantibody. A comparison of the immunoreactivity of mLL2 with that of cLL2in a competitive RIA showed that the two antibodies have nearlyidentical activities.

EXAMPLE 3 PCR/Gene Synthesis of the Humanized V Genes

The designed sequence for the hLL2 VH domain, the construction of thehLL2 VH domain by long oligonucleotides and PCR, and the staging vectorVHpBS containing the hLL.2 VH domain are summarized in the sketch shownin FIG. 6.

For the construction of the hLL2 VH domain, oligo A (149-mer) and oligoB (140-mer) were synthesized on an automated CYCLONE PLUS™ DNAsynthesizer (Milligen Bioresearch).

Oligo A (Sequence ID No. 10 below) represents the minus strand of thehLL2 VH domain complementary to nt 24 to 172.

    __________________________________________________________________________    Sequence ID No. 10                                                            __________________________________________________________________________    5'-TAT AAT CAT TCC TAG GAT TAA TGT ATC CAA TCC ATT CCA                        GAC CCT GTC CAG GTG CCT GCC TGA CCC AGT GCA GCC AGT AGC                       TAG TAA AGG TGT AGC CAG AAG CCT TGC AGG AGA CCT TCA CTG                       ATG ACC CAG GTT TCT TGA CTT CAG CC-3'                                         __________________________________________________________________________

Oligo B (Sequence ID No. 11 below) represents the minus strand of thehLL2 VH domain complementary to nt 180 to 320.

    __________________________________________________________________________    Sequence ID No. 11                                                            __________________________________________________________________________    5'-CCC CAG TAG AAC GTA GTA ATA TCC CTT CTT GCA CAA AAA TAA AAT                GCC GTG TCC TCA GAC CTC AGG CTG CTC AGC TCC ATC TAG GCT                       GTA TTG GTG GAT TCG TCT GCA GTT ATT GTG GCC TTG TCC TTG                       AAG TTC TGA TT-3'                                                             __________________________________________________________________________

Oligos A and B were cleaved from the support and deprotected bytreatment with concentrated ammonium hydroxide. After the samples werevacuum-dried (SpeedVac, Savant, Farmingdale, N.Y.) and resuspended in100 μl of water, incomplete oligomers (less than 100-mer) were removedby centrifugation through a CHROMOSPIN-100™ column (Clonetech, PaloAlto, Calif.) before the DNA oligomers were amplified by PCR. Allflanking primers for the separate amplifications and PCR cloning ofoligos A and B were purified by SDS-PAGE essentially according to themethods of Sambrook et al., 1989, above. From the CHROMASPIN-purifiedoligo A, 1 μl of sample stock was PCR-amplified in a reaction volume of100 μl by adding 5 μl of 5 μM of oligo Sequence ID No. 12:

    5'-CCA GCT GCA GCA ATC AGG GGC TGA AGT CAA GAA ACC TG-3'

and oligo Sequence ID No. 13:

    5'-AAG TGG ATC CTA TAA TCA TTC CTA GGA TTA ATG-3'

in the presence of 10 μl of 10×PCR Buffer (500 mM KCl, 100 mM Tris.HCLbuffer, pH 8.3, 15 mM MgCl₂) and 5 units of AMPLITAQ™ DNA polymerase(Perkin Elmer Cetus, Norwalk, Conn.). This reaction mixture wassubjected to 30 cycles of PCR reaction consisting of denaturation at 94°C. for 1 minute, annealing at 50° C. for 1.5 minutes, and polymerizationat 72° C. for 1.5 minutes.

Oligo B was PCR-amplified by the primer pairs Sequence ID No. 14:

    5'-TAA TCC TAG GAA TGA TTA TAC TGA GTA CAA TCA GAA CTT CAA GGA CCA G-3'

and Sequence ID No. 15:

    5'-GGA GAC GGT GAC CGT GGT GCC TTG GCC CCA GTA GAA CGT AGT AA-3'

under similar conditions.

Double-stranded PCR-amplified products for oligos A and B weregel-purified, restriction-digested with PstI/AvrII (PCR product of oligoA) and BstEII/AvrII (PCR product of oligo B), and subcloned into thecomplementary PstI/BstEII sites of the heavy chain staging vector,VHpBS. The humanized VH sequence was subcloned into the pG1g vector,resulting in the final human IgG1 heavy chain expression vector,hLL2pG1g.

For constructing the full length DNA of the humanized VK sequence, oligoE (150-mer) and oligo F (121-mer) were synthesized as described above.

Oligo E Sequence ID No. 16:

    __________________________________________________________________________    5'-CCT AGT GGA TGC CCA GTA GAT CAG CAG TTT AGG TGC TTT                        CCC TGG TTT CTG CTG GTA CCA GGC CAA GTA GTT CTT GTG ATT                       TGC ACT GTA TAA AAC ACT TTG ACT GGA CTT ACA GCT CAT AGT                       GAC CCT ATC TCC AAC AGA TGC GCT CAG-3'                                        __________________________________________________________________________

represents the minus strand of the humanized VK domain complementary tont 31 to 180, and this sequence was PCR-amplified by oligo Sequence IDNo. 17:

    5'-GAC AAG CTT CAG CTG ACC CAG TCT CCA TCA TCT CTG AGC GCA TCT GTT GGA G 3'

and oligo Sequence ID No. 18:

    5'-AGA GAA TCG CGA AGG GAC ACC AGA TTC CCT AGT GGA TGC CCA GTA-3'.

Oligo F Sequence ID No. 19:

    __________________________________________________________________________    5'- CCA GCT TGG TCC CTC CAC CGA ACG TCC ACG AGG AGA GGT                       ATT GGT GAC AAT AAT ATG TTG CAA TGT CTT CTG GTT GAA GAG                       AGC TGA TGG TGA AAG TAA AAT CTG TCC CAG ATC CGC TGC C-3'                      __________________________________________________________________________

represents the minus strand of the humanized LL2 VK domain complementaryto nt 208 to 327, and was PCR amplified by oligo Sequence ID No. 20:

    5'-GAC AAG CTT TCG CGA TTC TCT GGC AGC GGA TCT GGG ACA G-3'

and oligo Sequence ID No. 21:

    5'-GAC CGG CAG ATC TGC ACC TTG GTC CCT CCA CCG-3'.

Gel-purified PCR products for oligos E and F were restriction-digestedwith PvuII/NruI and NruI/BglIII, respectively. The two PCR fragments Eand F were then joined at the NruI site and ligated to the complementaryPvuI/BcII sites of the light chain staging vector, VKpBR. The humanizedVK sequence was subcloned into vector pKh to form the final , humankappa chain expression vector, hLL2pKh.

To express the humanized antibodies, about 10 μg of linearized hLL2pKhand 20 μg of linearized hLL2pG1g were used to transfect 5×10⁶ SP2/0cells by electroporation. The transfectomas were selected withhygromycin at 500 μg/ml and secreted antibody was purified on a 1×3 cmcolumn of protein A. After concentrating the purified antibody byCentricon 30 centrifugation, antibody concentration was determined byELISA. The final concentration of the antibody was adjusted to 1 mg/mlin PBS buffer containing 0.01% (w/v) sodium azide as a preservative.

In FIG. 1, there is compared the amino acid sequence between murine andhumanized LL2 VK domains (FIG. 1A, SEQ ID NOS. 2 and 6) and betweenmurine and humanized LL2 VH domains (FIG. 1B, SEQ ID NOS. 4, 9 and 8).In the VK chain, human REI framework sequences were used for all FRs. Inthe VH chain, human EU framework sequences were used for FR 1-3, andNEWM sequences were used for FR-4. Only human FR sequences that aredifferent from that of the mouse are shown. Asterisks indicate murine FRsequences that are different from that of the human FR at correspondingpositions. Murine residues at these positions were retained in thehumanized structure. CDRs are boxed.

In FIG. 4A (Sequence ID NOS. 1 and 2) there are shown the doublestranded DNA and corresponding amino acid sequences (shown by singleletter code) of the murine LL2 VK domain. CDR 1-3 amino acid sequencesare boxed. The corresponding display for VH is shown in FIG. 4B(Sequence ID NOS. 3 and 4).

In FIG. 5A (Sequence ID NOS. 5 and 6) and FIG. 5B (Sequence ID No. 7 and8) there are shown double-stranded DNA sequences and amino acidsequences of humanized LL2 VK and LL2 VH, respectively. Amino acidsequences are shown by the single-letter code, and CDR amino acidsequences are boxed.

EXAMPLE 4 Construction, Expression and Purification of Chimeric LL2Antibodies

The fragments containing the VK and VH sequences of LL2, together withthe promoter and signal peptide sequences, were excised from LL2VKpBRand LL2VHpBS, respectively, by double restriction digestion with HindIIIand BamHI. The about 600 bp VK fragments were then subcloned into theHindIII/BamHI site of a mammalian expression vector, pKh (FIG. 3A). pKhis a pSVhyg-based expression vector containing the genomic sequence ofthe human kappa constant region, an Ig enhancer, a kappa enhancer andthe hygromycin-resistant gene. Similarly, the ca. 800 bp VH fragmentswere subcloned into the corresponding HindIII/BamHI site of pG1g (FIG.3B), a pSVgpt-based expression vector carrying the genomic sequence ofthe human IgG1 constant region, an Ig enhancer and the xanthine-guaninephosphoribosyltransferase (gpt) gene. The final expression vectors aredesignated as LL2pKh and LL2pG1g, respectively.

The two plasmids were co-transfected into Sp2/0-Ag14 cells byelectroporation and selected for hygromycin resistance. Supernatantsfrom colonies surviving selection were monitored for chimeric antibodysecretion by ELISA assay (see above). The transfection efficiency wasapproximately 1-10×10⁶ cells. The antibody expression level, in aterminal culture, was found to vary in the range between <0.10 and2.5/μg/ml.

FIG. 7 shows the results of analyzing protein A-purified mLL2 (lanes 4and 7) and cLL2 (lanes 5 and 8) by SDS-PAGE under reducing andnonreducing conditions, respectively. HMW stands for high molecularweight protein markers, and LMW for light molecular weight markers. Thelight chains of both mLL2 and cLL2 (lanes 4 and 5) migrated primarily asa doublet band, with a higher than expected apparent molecular weight.As the human kappa constant region of cLL2 is known to contain nopotential glycosylation site, it can be inferred that the potentialglycosylation site identified in the FR1 region of LL2 VK domain wasutilized.

FIG. 8 shows the results of analyzing different versions of hLL2 andcLL2 antibodies by SDS-PAGE under reducing and non-reducing conditions.As before, LMW and HMW are molecular weight markers. Lanes 3 and 6 arecLL2 antibodies. Lanes 4 and 7 are hLL2 with seven murine FR residues inthe VH domain (hLL2-1). Lanes 5 and 8 are hLL2 with 6 murine FR residuesin the VH domain (hLL2-2). The humanized light chains migrated morerapidly and as more discrete bands compared to chimeric light chains.

FIG. 9 shows the results of SDS-PAGE analysis on mix-and-match and cLL2and hLL2 antibodies under both reducing and non-reducing conditions.Lanes 1 and 2 are molecular weight markers. Lanes 3 and 7 are cLL2.Lanes 4 and 8 are mix-and-match with a humanized light and chimericheavy chain (hL/cH)LL2!. Lanes 5 and 9 are chimeric light and humanizedheavy (Version 1) chains (cL/hH)LL2-1!. Lanes 6 and 10 are chimericlight and a humanized heavy (version 2) chains (cL/hH)LL2-2!. Thehumanized LL2 version 1 contains 7 murine FR residues in the VH domain,while version 2 contains 6 murine FR residues in the VH domain. It isnoteworthy that the position of the light chain of (hL/cH)LL2 (lane 4)is different from that of the others, suggesting that there is nocarbohydrate attachment to the humanized LL2 light chain.

EXAMPLE 5 Binding of cLL2 Antibody to Raji Cell Surface Antigens

A competition cell binding assay was carried out to assess theimmunoreactivity of cLL2 relative to the parent mLL2. Using ¹³¹I-labeled mLL2 (0.025 μg/ml) as a probe, Raji cells were incubated withthe antibodies and the relative binding to the cells determined from theamount of cell-bound labeled mLL2 (see above). As shown by thecompetition assays described in FIG. 10, both mLL2 and cLL2 antibodiesexhibited similar binding activities.

The results were confirmed by a second competition assay based on flowcytometry. Briefly, using Raji cells as before and varying theconcentration of one antibody relative to other, as before, the amountof bound mLL2 or cLL2 was determined with FITC-labeled anti-mouse Fc oranti-human Fc antibodies followed by analysis using flow cytometry.

EXAMPLE 6 Binding of hLL2 Antibodies to Raji Cells

In experiments similar to those of Example 5, the antigen bindingaffinities of the three different combinations of mix-and-match orhumanized LL2 were compared with that of cLL2 in the flow cytometryassay.

Briefly, 1 μg of cLL2, mix-and-match LL2, hLL2-1 or hLL2-2 antibodieswere incubated with 10⁸ Raji cells in the presence of varyingconcentrations of mLL2 F(ab')₂ fragments (as competitor) in a finalvolume of 100 μl of PBS buffer supplemented with 1% FCS and 0.01% sodiumazide. The mixture was incubated for 30 minutes at 4° C., and washedthree times with PBS to remove unbound antibodies. By taking advantageof the presence of human Fc portions in the antibodies, the bindinglevels of the antibodies were assessed by adding a 20× dilutedFITC-labeled goat anti-human IgG1, Fc fragment-specific antibodies(Jackson ImmunoResearch, West Grove, Pa.). The cells were washed threetimes with PBS, and fluorescence intensities measured by a FACSCANfluorescence activated cell sorter (Becton-Dickinson, Bedford, Mass.).The results are shown in FIG. 11A.

Using the same methods, cLL2 was compared to two versions of hLL2 (FIG.11B).

The results shown in FIG. 11A and B demonstrate that theimmunoreactivity of cLL2 is similar or identical to that of humanized ormix-and-match antibodies. Taken together with the comparison of cLL2with mLL2 (FIG. 10), the authenticity of the sequences for chimeric andhumanized VK and VH obtained is established, and the functionality ofcLL2 and hLL2 confirmed.

EXAMPLE 7 Internalization of mLL2 and cLL2 by Raji Cells

One of the unique characteristics of the LL2 antibody is its rapidinternalization upon binding to Raji cells (Shih et al., 1994 above).Murine LL2 after internalization is likely to be rapidly transferred tothe Golgi apparatus and from there to the lysosomes, the organelleresponsible for the degradation of a wide variety of biochemicals(Keisari et al., Immunochem., 10:565 (1973)).

Rates of antibody internalization were determined according to Opreskoet al., 1987 above. The ratio of CPM_(intracellular) /CPM_(surface) wasdetermined as a function of time.

Rates of LL2 antibody internalization were determined by incubatingradiolabeled LL2 antibody (1×10⁶ cpm) with 0.5×10⁶ Raji cells in 0.5 mlof DMEM buffer containing 1% human serum for 2 hrs. at 4° C. Excesshuman serum was included to saturate Raji cell surface Fc receptors inorder to exclude or minimize non-antigen-specific internalizationmediated through the Fc receptors. Unbound radiolabeled LL2 antibodieswere removed from the cells by washing three times with 0.5 ml portionsof DMEM at 4° C. Cells were then incubated at 37° C., and, at timedintervals, aliquots of the cell suspension were transferred to ice inorder to stop internalization. The cells in these aliquots were isolatedby centrifugation at 1,000×g for 5 mins. at 4° C., and surface boundradiolabeled LL2 stripped off cells with 1 ml of 0.1M glycine acetatebuffer, pH 3, for 8 mins. at 4° C. Radioactivity thus obtained (CPMsurface) and radioactivity remaining in the cells (CPM intracellular)were determined. Rates of internalization were calculated from the slopeof the plot of intracellular:surface radioactivity ratios as a functionof time.

As shown in FIG. 12, mLL2, cLL2, cLL2Q and hLL2 antibodies wereinternalized at a similar rate (Ke=0.107 (mLL2) to 0.1221 (cLL2Q, NVT toQVT mutation). Those numbers suggested that approximately 50% of thesurface-bound antibody could be internalized in 10 min. The results showthat neither chimerization nor humanization nor deglycosylation bymutagenesis of mLL2 antibodies impair rates of internalization.

The pattern of internalization for mLL2, cLL2 and hLL2 was alsomonitored by fluorescence microscopy on a time-course basis using aFITC-labeled second antibody probe as described in the specification.Internalization of both antibodies was observed in at the earliest timepoint measurable. At 5 minutes, antibodies were seen both on the cellsurface and internalized in areas immediately adjacent to the membraneas cytoplasmic micro-vesicles. At 15 min. post-incubation, the fine dotsdispersed around the intramembrane began to merge into a group ofgranules, at locations believed to be the Golgi apparatus. As moreantibodies were being internalized after 30 min. of incubation,redistribution of the grouped antibodies to scattered locations,probably the lysosomes in which the antibodies were degraded, wasobserved. At 2 hrs post-incubation, most of the antibodies were foundinside the cell. Only strong surface staining was observed when LL2 wasincubated for 20 min on ice. Both mLL2 and cLL2 were internalized with asimilar pattern. The internalization of LL2 was associated specificallywith antigen-antibody binding, as the irrelevant control humanizedantibody demonstrated only dull surface staining.

A103 antibody (an IgG2a antibody that binds to the surface of all humanepithelial cells but does not internalize efficiently (Mattes et al.,Hybridoma, 2:253 (1983)) showed strong membrane staining at up to 2 h,while the anti-transferrin receptor antibody (5F9) internalized rapidly,just as did LL2.

EXAMPLE 8 Role of Glycosylation Site in FR1 Region of LL2 VK Sequence

Of particular inventive interest is the identification of anAsn-glycosylation site at position 18-20 within the FR1 region of theLL2 NVT light chain sequence (FIG. 4A SEQ ID NO. 2). As shown above,SDS-PAGE analysis under reducing condition suggests that the Asnglycosylation site is utilized for carbohydrate addition.

In this example, the influence of the carbohydrate moiety at position18-20 on the functional activities of the light chains was examined.

Murine and chimeric LL2 light chains were treated with (+) or without(-) endoglycosidase F conventionally, and the antibody products examinedby SDS-PAGE under reducing and non-reducing conditions (FIG. 13). Therewas no distinction between the antibody types as to electrophoreticbehavior. In both cases, deglycosylation reduced the rate of migrationof the light chain.

The effect of deglycosylation on the binding affinity to Raji cells ofthe mLL2 antibody is shown in FIG. 14. Removing carbohydrate byendoglycosidase F was without influence on the binding activity.

A mutation was introduced at position 18 of the light chain so that theAsn was replaced with Gln to produce LL2Q VK FR1. SDS-PAGE analysesdemonstrated that the NVT to QVT mutation abolished glycosylation of theantibody. Comparison of the Raji cell binding affinity for cLL2 with andwithout light chain VK glycosylation demonstrated that the carbohydratemoiety was without influence on binding of the antibody to these cells.

It can be concluded that the presence of the carbohydrate site in thevariable region does not affect the immunoreactivity of the antibody.Computer modeling studies suggested that the VK carbohydrate moiety inLL2 is remotely positioned from the CDRs and forms a "cap" over thebottom loops of the FR-associated β-barrels supporting the CDRs.

Humanization without inclusion of the original glycosylation siteresulted in a CDR-grafted LL2 antibody with immunoreactivity comparableto that of its murine counterpart.

These characteristics indicate that the glycosylation site can be usedfor conjugating therapeutic or diagnostic agents to LL2 withoutcompromising the ability of the antibody to bind and internalize inB-lymphoma or leukemia cells.

EXAMPLE 9 Conjugation of LL2 at its Carbohydrate-bearing Site

The apparent lack of involvement of the variable region carbohydratemoiety in the functional activities of mLL2, cLL2 and hLL2 mAbsindicates that this moiety could profitably be used as the site ofattachment of cytotoxic or detection agents such as radionuclides ortoxins, and thereby avoid potential interference with the binding of theconjugate to a cell surface.

Using procedures described in Shih et al., U.S. Pat. No. 5,057,313(which is incorporated by reference) for preparing antibody conjugatesthrough an oxidized carbohydrate moiety of the antibody and a primaryalkylamino group of a polymeric carrier to which are covalently one ormore of a variety of drugs, toxins, chelators and detectable labels, adoxorubicin-dextran-LL2 antibody fragment devoid of appended glycans wasproduced containing multiple copies of the drug. The carbohydratemoieties of the cLL2 VK FR1 region involved were those covalently boundto the Asn glycosylation site.

In one synthesis, dextran (18-40 kDa) was converted to an amino dextranby oxidation of the dextran by NaIO₄, Schiff base formation with NH₂--CH₂ --CHOH--CH₂ --NH₂, and reduction with NaBH₄. The amino dextran wasthen condensed with doxorubicin (DOX) in the presence of succinicanhydride and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to produceDOX-aminodextran. The latter was then condensed with an aldehydic groupon LL2 VK FR-1 produced by oxidizing the carbohydrate moiety of theantibody fragment with NaIO₄.

In one preparation of DOX-LL2, the number of moles of DOX attached todextran was 14 moles per mole dextran, and the number of moles ofdoxorubicin per mole F(ab')2 was 8.9. The immunoreactivity in the Rajicell binding assay above was about 80% of control values.

This conjugation system is not limited to the mLL2 antibody. In acomparative study, 15-19 moles of DOX/mole of cLL2 were bound.

The conjugation possibilities are not limited to the use of a carrierdextran as in the example above. For example, the carbohydrate moiety ofthe LL2 VK FR1 region can be oxidized to produce aldehydic groups. Thesein turn can be reacted with an amino group on any drug to produce aSchiff base which, upon reduction, produces multiple copies of the drugstably linked to the antibody via alkyamine groups.

For example, where the drug is aminohexyl DTPA (a chelating agent),there is produced a LL2 covalently bound to a chelator. The chelator canbe used to deliver to target tissues, for example, a radionuclide orparamagnetic metal ion, with a potential for diagnostic and therapeuticuses. DTPA-LL2 conjugates were produced containing 5.5 moles of thechelator/mole of antibody which, in turn, chelated 47.3% of Y-90 and97.4% In-111

It should be emphasized that the above-described examples merelydescribe several specific embodiments of the invention, and applicantsdo not intend to be limited as to scope of claims by these specificexamples.

Applicants also incorporate by reference all publications and patentscited in the specification.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 339 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..339                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GACATTCAGCTGACCCAGTCTCCATCATCTCTGGCTGTGTCTGCAGGA48                            AspIleGlnLeuThrGlnSerProSerSerLeuAlaValSerAlaGly                              151015                                                                        GAAAACGTCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGT96                            GluAsnValThrMetSerCysLysSerSerGlnSerValLeuTyrSer                              202530                                                                        GCAAATCACAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAG144                           AlaAsnHisLysAsnTyrLeuAlaTrpTyrGlnGlnLysProGlyGln                              354045                                                                        TCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTC192                           SerProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal                              505560                                                                        CCTGATCGCTTCACAGGCAGCGGATCTGGGACAGATTTTACTCTTACC240                           ProAspArgPheThrGlySerGlySerGlyThrAspPheThrLeuThr                              65707580                                                                      ATCAGCAGAGTACAAGTTGAAGACCTGGCAATTTATTATTGTCACCAA288                           IleSerArgValGlnValGluAspLeuAlaIleTyrTyrCysHisGln                              859095                                                                        TACCTCTCCTCGTGGACGTTCGGTGGAGGGACCAAGCTGGAGATCAAA336                           TyrLeuSerSerTrpThrPheGlyGlyGlyThrLysLeuGluIleLys                              100105110                                                                     CGT339                                                                        Arg                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspIleGlnLeuThrGlnSerProSerSerLeuAlaValSerAlaGly                              151015                                                                        GluAsnValThrMetSerCysLysSerSerGlnSerValLeuTyrSer                              202530                                                                        AlaAsnHisLysAsnTyrLeuAlaTrpTyrGlnGlnLysProGlyGln                              354045                                                                        SerProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal                              505560                                                                        ProAspArgPheThrGlySerGlySerGlyThrAspPheThrLeuThr                              65707580                                                                      IleSerArgValGlnValGluAspLeuAlaIleTyrTyrCysHisGln                              859095                                                                        TyrLeuSerSerTrpThrPheGlyGlyGlyThrLysLeuGluIleLys                              100105110                                                                     Arg                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 348 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..348                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAGGTCCAGCTGCAGGAGTCAGGGGCTGAACTGTCAAAACCTGGGGCC48                            GlnValGlnLeuGlnGluSerGlyAlaGluLeuSerLysProGlyAla                              151015                                                                        TCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTAC96                            SerValLysMetSerCysLysAlaSerGlyTyrThrPheThrSerTyr                              202530                                                                        TGGCTGCACTGGATAAAACAGAGGCCTGGACAGGGTCTGGAATGGATT144                           TrpLeuHisTrpIleLysGlnArgProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GGATACATTAATCCTAGGAATGATTATACTGAGTACAATCAGAACTTC192                           GlyTyrIleAsnProArgAsnAspTyrThrGluTyrAsnGlnAsnPhe                              505560                                                                        AAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTAC240                           LysAspLysAlaThrLeuThrAlaAspLysSerSerSerThrAlaTyr                              65707580                                                                      ATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGT288                           MetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCys                              859095                                                                        GCAAGAAGGGATATTACTACGTTCTACTGGGGCCAAGGCACCACTCTC336                           AlaArgArgAspIleThrThrPheTyrTrpGlyGlnGlyThrThrLeu                              100105110                                                                     ACAGTCTCCTCG348                                                               ThrValSerSer                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlnValGlnLeuGlnGluSerGlyAlaGluLeuSerLysProGlyAla                              151015                                                                        SerValLysMetSerCysLysAlaSerGlyTyrThrPheThrSerTyr                              202530                                                                        TrpLeuHisTrpIleLysGlnArgProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GlyTyrIleAsnProArgAsnAspTyrThrGluTyrAsnGlnAsnPhe                              505560                                                                        LysAspLysAlaThrLeuThrAlaAspLysSerSerSerThrAlaTyr                              65707580                                                                      MetGlnLeuSerSerLeuThrSerGluAspSerAlaValTyrTyrCys                              859095                                                                        AlaArgArgAspIleThrThrPheTyrTrpGlyGlnGlyThrThrLeu                              100105110                                                                     ThrValSerSer                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 339 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..339                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GACATTCAGCTGACCCAGTCTCCATCATCTCTGAGCGCATCTGTTGGA48                            AspIleGlnLeuThrGlnSerProSerSerLeuSerAlaSerValGly                              151015                                                                        GATAGGGTCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTATACAGT96                            AspArgValThrMetSerCysLysSerSerGlnSerValLeuTyrSer                              202530                                                                        GCAAATCACAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGAAA144                           AlaAsnHisLysAsnTyrLeuAlaTrpTyrGlnGlnLysProGlyLys                              354045                                                                        GCACCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTC192                           AlaProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal                              505560                                                                        CCTTCGCGATTCTCTGGCAGCGGATCTGGGACAGATTTTACTTTCACC240                           ProSerArgPheSerGlySerGlySerGlyThrAspPheThrPheThr                              65707580                                                                      ATCAGCTCTCTTCAACCAGAAGACATTGCAACATATTATTGTCACCAA288                           IleSerSerLeuGlnProGluAspIleAlaThrTyrTyrCysHisGln                              859095                                                                        TACCTCTCCTCGTGGACGTTCGGTGGAGGGACCAAGCTGGAGATCAAA336                           TyrLeuSerSerTrpThrPheGlyGlyGlyThrLysLeuGluIleLys                              100105110                                                                     CGT339                                                                        Arg                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 113 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AspIleGlnLeuThrGlnSerProSerSerLeuSerAlaSerValGly                              151015                                                                        AspArgValThrMetSerCysLysSerSerGlnSerValLeuTyrSer                              202530                                                                        AlaAsnHisLysAsnTyrLeuAlaTrpTyrGlnGlnLysProGlyLys                              354045                                                                        AlaProLysLeuLeuIleTyrTrpAlaSerThrArgGluSerGlyVal                              505560                                                                        ProSerArgPheSerGlySerGlySerGlyThrAspPheThrPheThr                              65707580                                                                      IleSerSerLeuGlnProGluAspIleAlaThrTyrTyrCysHisGln                              859095                                                                        TyrLeuSerSerTrpThrPheGlyGlyGlyThrLysLeuGluIleLys                              100105110                                                                     Arg                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 348 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..348                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CAGGTCCAGCTGGTCCAATCAGGGGCTGAAGTCAAGAAACCTGGGTCA48                            GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlySer                              151015                                                                        TCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTAC96                            SerValLysValSerCysLysAlaSerGlyTyrThrPheThrSerTyr                              202530                                                                        TGGCTGCACTGGGTCAGGCAGGCACCTGGACAGGGTCTGGAATGGATT144                           TrpLeuHisTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GGATACATTAATCCTAGGAATGATTATACTGAGTACAATCAGAACTTC192                           GlyTyrIleAsnProArgAsnAspTyrThrGluTyrAsnGlnAsnPhe                              505560                                                                        AAGGACAAGGCCACAATAACTGCAGACGAATCCACCAATACAGCCTAC240                           LysAspLysAlaThrIleThrAlaAspGluSerThrAsnThrAlaTyr                              65707580                                                                      ATGGAGCTGAGCAGCCTGAGGTCTGAGGACACGGCATTTTATTTTTGT288                           MetGluLeuSerSerLeuArgSerGluAspThrAlaPheTyrPheCys                              859095                                                                        GCAAGAAGGGATATTACTACGTTCTACTGGGGCCAAGGCACCACGGTC336                           AlaArgArgAspIleThrThrPheTyrTrpGlyGlnGlyThrThrVal                              100105110                                                                     ACCGTCTCCTCG348                                                               ThrValSerSer                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlySer                              151015                                                                        SerValLysValSerCysLysAlaSerGlyTyrThrPheThrSerTyr                              202530                                                                        TrpLeuHisTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GlyTyrIleAsnProArgAsnAspTyrThrGluTyrAsnGlnAsnPhe                              505560                                                                        LysAspLysAlaThrIleThrAlaAspGluSerThrAsnThrAlaTyr                              65707580                                                                      MetGluLeuSerSerLeuArgSerGluAspThrAlaPheTyrPheCys                              859095                                                                        AlaArgArgAspIleThrThrPheTyrTrpGlyGlnGlyThrThrVal                              100105110                                                                     ThrValSerSer                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 116 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlnValGlnLeuGlnGlnSerGlyAlaGluValLysLysProGlySer                              151015                                                                        SerValLysValSerCysLysAlaSerGlyTyrThrPheThrSerTyr                              202530                                                                        TrpLeuHisTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpIle                              354045                                                                        GlyTyrIleAsnProArgAsnAspTyrThrGluTyrAsnGlnAsnPhe                              505560                                                                        LysAspLysAlaThrIleThrAlaAspGluSerThrAsnThrAlaTyr                              65707580                                                                      MetGluLeuSerSerLeuArgSerGluAspThrAlaPheTyrPheCys                              859095                                                                        AlaArgArgAspIleThrThrPheTyrTrpGlyGlnGlyThrThrVal                              100105110                                                                     ThrValSerSer                                                                  115                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 149 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TATAATCATTCCTAGGATTAATGTATCCAATCCATTCCAGACCCTGTCCAGGTGCCTGCC60                TGACCCAGTGCAGCCAGTAGCTAGTAAAGGTGTAGCCAGAAGCCTTGCAGGAGACCTTCA120               CTGATGACCCAGGTTTCTTGACTTCAGCC149                                              (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 140 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCCCAGTAGAACGTAGTAATATCCCTTCTTGCACAAAAATAAAATGCCGTGTCCTCAGAC60                CTCAGGCTGCTCAGCTCCATGTAGGCTGTATTGGTGGATTCGTCTGCAGTTATTGTGGCC120               TTGTCCTTGAAGTTCTGATT140                                                       (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CCAGCTGCAGCAATCAGGGGCTGAAGTCAAGAAACCTG38                                      (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AAGTGGATCCTATAATCATTCCTAGGATTAATG33                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TAATCCTAGGAATGATTATACTGAGTACAATCAGAACTTCAAGGACCAG49                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GGAGACGGTGACCGTGGTGCCTTGGCCCCAGTAGAACGTAGTAA44                                (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CCTAGTGGATGCCCAGTAGATCAGCAGTTTAGGTGCTTTCCCTGGTTTCTGCTGGTACCA60                GGCCAAGTAGTTCTTGTGATTTGCACTGTATAAAACACTTTGACTGGACTTACAGCTCAT120               AGTGACCCTATCTCCAACAGATGCGCTCAG150                                             (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GACAAGCTTCAGCTGACCCAGTCTCCATCATCTCTGAGCGCATCTGTTGGAG52                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AGAGAATCGCGAAGGGACACCAGATTCCCTAGTGGATGCCCAGTA45                               (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CCAGCTTGGTCCCTCCACCGAACGTCCACGAGGAGAGGTATTGGTGACAATAATATGTTG60                CAATGTCTTCTGGTTGAAGAGAGCTGATGGTGAAAGTAAAATCTGTCCCAGATCCGCTGC120               C121                                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GACAAGCTTTCGCGATTCTCTGGCAGCGGATCTGGGACAG40                                    (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GACCGGCAGATCTGCACCTTGGTCCCTCCACCG33                                           __________________________________________________________________________

We claim:
 1. A humanized LL2 (hLL2) monoclonal antibody, (mAb),designated hLL2-2, comprising the complementarity-determining regions(CDRs) of mouse LL2 (mLL2), wherein said hLL2-2 mAb retainssubstantially the B-lymphoma cell and leukemia cell targeting and cellinternalization characteristics of said mLL2 mAb, and wherein saidhLL2-2 mAb is less immunogenic in a human subject than is said mLL2 mAb,and wherein said hLL2-2 mAb comprises the heavy chain variable region ofSEQ ID NO: 8 and the light chain variable region of SEQ ID NO:
 6. 2. AB-lymphoma cell and leukemia cell targeting conjugate, comprising thehLL2-2 mAb of claim 1 or a specific binding fragment thereof that bindsto said cells, wherein said hLL2-2 mAb or said specific binding fragmentthereof is covalently bound to a diagnostic or therapeutic reagent.
 3. Aconjugate according to claim 2 wherein said diagnostic reagent comprisesa label.
 4. A conjugate according to claim 2 wherein said therapeuticreagent comprises a cytotoxic agent.
 5. A conjugate according to claim2, wherein said reagent is bound to said mAb, or specific cell bindingfragment thereof, by means of a carbohydrate moiety of said mAb or cellbinding fragment thereof.
 6. The conjugate according, to claim 4,wherein said cytotoxic agent is a toxin.
 7. A method of treating aB-cell lymphoma cell or leukemia cell in a subject, comprising the stepof administering to said subject a therapeutically effective amount ofthe conjugate according to claim 4 formulated in a pharmaceuticallyacceptable vehicle.
 8. A method of diagnosing a B-cell lymphoma cell orleukemia cell in a subject, comprising the steps of administering tosaid subject a diagnostically effective amount of the conjugateaccording to claim 3 formulated in a pharmaceutically acceptablevehicle, and detecting said label.
 9. A humanized LL2 (hLL2) monoclonalantibody, (mAb), designated hLL2-1, comprising thecomplementarity-determining regions (CDRs) of mouse LL2 (mLL2), whereinsaid hLL2-1 mAb retains substantially the B-lymphoma cell and leukemiacell targeting and cell internalization characteristics of said mLL2mAb, and wherein said hLL2-1 mAb is less immunogenic in a human subjectthan is said mLL2 mAb, and wherein said hLL2-1 mAb comprises the heavychain variable region of SEQ ID NO: 9 and the light chain variableregion of SEQ ID NO:
 6. 10. A B-lymphoma cell and leukemia celltargeting conjugate, comprising the hLL2-1 mAb of claim 9 or a specificbinding fragment thereof that binds to said cells, wherein said hLL2-1mAb or said specific binding fragment thereof is covalently bound to adiagnostic or therapeutic reagent.
 11. A conjugate according to claim10, wherein said diagnostic reagent comprises a label.
 12. A conjugateaccording to claim 10, wherein said therapeutic reagent comprises acytotoxic agent.
 13. The conjugate according to claim 12, wherein saidcytotoxic agent is a toxin.
 14. A conjugate according to claim 10,wherein said reagent is bound to said mAb, or specific cell bindingfragment thereof, by means of a carbohydrate moiety of said mAb or cellbinding fragment thereof.
 15. A method of treating a B-cell lymphomacell or leukemia cell in a subject, comprising the step of administeringto said subject a therapeutically effective amount of the conjugateaccording to claim 12, formulated in a pharmaceutically acceptablevehicle.
 16. A method of diagnosing a B-cell lymphoma cell or leukemiacell in a subject, comprising the steps of administering to said subjecta diagnostically effective amount of the conjugate according to claim11, formulated in a pharmaceutically acceptable vehicle, and detectingsaid label.
 17. An expression vector comprising a DNA sequence encodingthe humanized hLL2-2 heavy chain variable region of SEQ ID NO:
 8. 18.The expression vector of claim 17, wherein said vector further comprisesan Ig promoter, a DNA sequence encoding a signal peptide, a genomicsequence encoding a human IgG1 heavy chain constant region, an Igenhancer and at least one DNA sequence encoding a marker.
 19. Theexpression vector of claim 17, wherein said DNA sequence is SEQ ID NO:7.
 20. The humanized LL2-2 heavy chain variable region of SEQ ID NO: 8.21. A humanized LL2 heavy chain comprising the heavy chain variableregion of claim
 20. 22. An expression vector comprising a DNA sequenceencoding the humanized hLL2 light chain variable region of SEQ ID NO: 6.23. The expression vector of claim 22 wherein said vector furthercomprises an Ig promoter, a DNA sequence encoding a signal peptide, agenomic sequence encoding a human kappa light chain constant region, ankappa enhancer and at least one DNA sequence encoding a marker.
 24. Theexpression vector of claim 22, wherein said DNA sequence is SEQ ID NO:5.
 25. The humanized LL2 light chain variable region of SEQ ID NO: 6.26. A humanized LL2 light chain comprising the light chain variableregion of claim
 25. 27. An expression vector comprising a DNA sequenceencoding the humanized hLL2-1 heavy chain variable region of SEQ ID NO:9.
 28. The vector of claim 27, wherein said vector further comprises anIg promoter, a DNA sequence encoding a signal peptide, a genomicsequence encoding a human IgG1 heavy chain constant region, an Igenhancer and at least one DNA sequence encoding a marker.
 29. Thehumanized LL2-1 heavy chain variable region of SEQ ID NO:9.
 30. Ahumanized LL2 heavy chain comprising the heavy chain variable region ofclaim
 29. 31. A method for the expression of a humanized LL2 monoclonalantibody, designated hLL2-2, comprising:(a) linearizing the expressionvector of claim 17 and an expression vector comprising a DNA sequenceencoding the humanized hLL2 light chain variable region of SEQ ID NO: 6;(b) transfecting mammalian cells with said linearized vectors; (c)selecting said transfected cells which express a xanthine-guaninephosphoribosyltransferase (gpt) gene; and (d) selecting cells secretingsaid humanized monoclonal antibody from said cells which express saidgpt gene.
 32. A method for the expression of a humanized LL2 monoclonalantibody, designated hLL2-1, comprising:(a) linearizing the expressionvector of claim 27 and an expression vector comprising a DNA sequenceencoding the humanized hLL2 light chain variable region of SEQ ID NO: 6;(b) transfecting mammalian lymphoma cells with said linearized vectors;(c) selecting said transfected cells which express the xanthine-guaninephosphoribosyltransferase (gpt) gene; and (d) selecting cells secretingsaid humanized monoclonal antibody from said cells which express saidgpt gene.